Proteasome inhibition potentiates Kv1.3 potassium channel expression as therapeutic target in drug-sensitive and -resistant human melanoma cells.

Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches. Here, we investigated the combination of proteasome and Kv1.3 potassium channel inhibition on chemo-resistant, BRAF inhibitor-resistant as well as sensitive human melanoma cells. Our experiments demonstrated that all analyzed melanoma cell lines were sensitive to proteasome inhibitor treatment at concentrations that are not toxic to primary human fibroblasts. To further reduce proteasome inhibitor-associated side effects, and to foster apoptosis, potassium channels, which are other targets to induce pro-apoptotic effects in cancer cells, were blocked. In support, combined exposure of melanoma cells to proteasome and Kv1.3 channel inhibitor resulted in synergistic effects and significantly reduced cell viability. On the molecular level, enhanced apoptosis correlated with an increase of intracellular Kv1.3 channels and pro-apoptotic proteins such as Noxa and Bak and a reduction of anti-apoptotic proteins. Thus, use of combined therapeutic strategies triggering different apoptotic pathways may efficiently prevent the outgrowth of drug-resistant and -sensitive BRAF-mutant melanoma cells. In addition, this could be the basis for an alternative approach to treat other tumors expressing mutated BRAF such as non-small-cell lung cancer.

Spatially resolved protein map of intact human cytomegalovirus virions.

Herpesviruses assemble large enveloped particles that are difficult to characterize structurally due to their size, fragility and complex multilayered proteome with partially amorphous nature. Here we used crosslinking mass spectrometry and quantitative proteomics to derive a spatially resolved interactome map of intact human cytomegalovirus virions. This enabled the de novo allocation of 32 viral proteins into four spatially resolved virion layers, each organized by a dominant viral scaffold protein. The viral protein UL32 engages with all layers in an N-to-C-terminal radial orientation, bridging nucleocapsid to viral envelope. We observed the layer-specific incorporation of 82 host proteins, of which 39 are selectively recruited. We uncovered how UL32, by recruitment of PP-1 phosphatase, antagonizes binding to 14-3-3 proteins. This mechanism assures effective viral biogenesis, suggesting a perturbing role of UL32-14-3-3 interaction. Finally, we integrated these data into a coarse-grained model to provide global insights into the native configuration of virus and host protein interactions inside herpesvirions.

Abemaciclib restricts HCMV replication by suppressing pUL97-mediated phosphorylation of SAMHD1.

Human cytomegalovirus (HCMV) is a herpesvirus that causes life-threatening infections in newborns or immunosuppressed patients. For viral replication, HCMV establishes a network of cellular interactions, among others cyclin-dependent kinases (CDK). Furthermore, HCMV encodes pUL97, a viral kinase, which is a CDK-homologue. HCMV uses pUL97 in order to phosphorylate and thereby antagonize SAMHD1, an antiviral host cell factor. Since HCMV has several mechanisms to evade restriction by SAMHD1, we first analyzed the kinetics of SAMHD1-inactivation and found that phosphorylation of SAMHD1 by pUL97 occurs directly after infection of macrophages. We hence hypothesized that inhibition of this process qualifies as efficient antiviral target and FDA approved CDK-inhibitors (CDKIs) might be potent antivirals that prevent the inactivation of SAMHD1. Indeed, Abemaciclib, a 2nd generation CDKI exhibited superior IC50s against HCMV in infected macrophages and the antiviral activity largely relied on its ability to block pUL97-mediated SAMHD1-phosphorylation. Altogether, our study highlights the therapeutic potential of clinically-approved CDKIs as antivirals against HCMV, sheds light on their mode of action and establishes SAMHD1 as a valid and highly potent therapeutic target.

Engineering, decoding and systems-level characterization of chimpanzee cytomegalovirus.

The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious viruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci and a deletion-prone region within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the viral transcriptome and proteome and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMV's cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV.

Emerging Mechanisms of G1/S Cell Cycle Control by Human and Mouse Cytomegaloviruses.

Cytomegaloviruses (CMVs) are among the largest pathogenic viruses in mammals. To enable replication of their long double-stranded DNA genomes, CMVs induce profound changes in cell cycle regulation. A hallmark of CMV cell cycle control is the establishment of an unusual cell cycle arrest at the G1/S transition, which is characterized by the coexistence of cell cycle stimulatory and inhibitory activities. While CMVs interfere with cellular DNA synthesis and cell division, they activate S-phase-specific gene expression and nucleotide metabolism. This is facilitated by a set of CMV gene products that target master regulators of G1/S progression such as cyclin E and A kinases, Rb-E2F transcription factors, p53-p21 checkpoint proteins, the APC/C ubiquitin ligase, and the nucleotide hydrolase SAMHD1. While the major themes of cell cycle regulation are well conserved between human and murine CMVs (HCMV and MCMV), there are considerable differences at the level of viral cell cycle effectors and their mechanisms of action. Furthermore, both viruses have evolved unique mechanisms to sense the host cell cycle state and modulate the infection program accordingly. This review provides an overview of conserved and divergent features of G1/S control by MCMV and HCMV.

Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.

Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of ß-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.

Murine Cytomegalovirus M25 Proteins Sequester the Tumor Suppressor Protein p53 in Nuclear Accumulations.

To ensure productive infection, herpesviruses utilize tegument proteins and nonstructural regulatory proteins to counteract cellular defense mechanisms and to reprogram cellular pathways. The M25 proteins of mouse cytomegalovirus (MCMV) belong to the betaherpesvirus UL25 gene family that encodes viral proteins implicated with regulatory functions. Through affinity purification and mass spectrometric analysis, we discovered the tumor suppressor protein p53 as a host factor interacting with the M25 proteins. M25-p53 interaction in infected and transfected cells was confirmed by coimmunoprecipitation. Moreover, the proteins colocalized in nuclear dot-like structures upon both infection and inducible expression of the two M25 isoforms. p53 accumulated in wild-type MCMV-infected cells, while this did not occur upon infection with a mutant lacking the M25 gene. Both M25 proteins were able to mediate the effect, identifying them as the first CMV proteins responsible for p53 accumulation during infection. Interaction with M25 proteins led to substantial prolongation of the half-life of p53. In contrast to the higher abundance of the p53 protein in wild-type MCMV-infected cells, the transcript levels of the prominent p53 target genes Cdkn1a and Mdm2 were diminished compared to cells infected with the ΔM25 mutant, and this was associated with reduced binding of p53 to responsive elements within the respective promoters. Notably, the productivity of the M25 deletion mutant was partially rescued on p53-negative fibroblasts. We propose that the MCMV M25 proteins sequester p53 molecules in the nucleus of infected cells, reducing their availability for activating a subset of p53-regulated genes, thereby dampening the antiviral role of p53.IMPORTANCE Host cells use a number of factors to defend against viral infection. Viruses are, however, in an arms race with their host cells to overcome these defense mechanisms. The tumor suppressor protein p53 is an important sensor of cell stress induced by oncogenic insults or viral infections, which upon activation induces various pathways to ensure the integrity of cells. Viruses have to counteract many functions of p53, but complex DNA viruses such as cytomegaloviruses may also utilize some p53 functions for their own benefit. In this study, we discovered that the M25 proteins of mouse cytomegalovirus interact with p53 and mediate its accumulation during infection. Interaction with the M25 proteins sequesters p53 molecules in nuclear dot-like structures, limiting their availability for activation of a subset of p53-regulated target genes. Understanding the interaction between viral proteins and p53 may allow to develop new therapeutic strategies against cytomegalovirus and other viruses.

The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant.

Pandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular basis of this species barrier is incompletely understood. Here we combine metabolic pulse labeling and quantitative proteomics to monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain and observe striking differences in viral protein synthesis. Most importantly, the matrix protein M1 is inefficiently produced by the bird-adapted strain. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Strain-specific M segment splicing is controlled by the 3' splice site and functionally important for permissive infection. In silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, we identify M segment RNA splicing as a viral host range determinant.

Human cytomegalovirus overcomes SAMHD1 restriction in macrophages via pUL97.

The host restriction factor sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is an important component of the innate immune system. By regulating the intracellular nucleotide pool, SAMHD1 influences cell division and restricts the replication of viruses that depend on high nucleotide concentrations. Human cytomegalovirus (HCMV) is a pathogenic virus with a tropism for non-dividing myeloid cells, in which SAMHD1 is catalytically active. Here we investigate how HCMV achieves efficient propagation in these cells despite the SAMHD1-mediated dNTP depletion. Our analysis reveals that SAMHD1 has the capability to suppress HCMV replication. However, HCMV has evolved potent countermeasures to circumvent this block. HCMV interferes with SAMHD1 steady-state expression and actively induces SAMHD1 phosphorylation using the viral kinase pUL97 and by hijacking cellular kinases. These actions convert SAMHD1 to its inactive phosphorylated form. This mechanism of SAMHD1 inactivation by phosphorylation might also be used by other viruses to overcome intrinsic immunity.

A viral kinase counteracts in vivo restriction of murine cytomegalovirus by SAMHD1.

The deoxynucleotide triphosphate (dNTP) hydrolase SAMHD1 inhibits retroviruses in non-dividing myeloid cells. Although antiviral activity towards DNA viruses has also been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains unclear. To determine the impact of SAMHD1 on the replication of CMV, we used murine CMV (MCMV) to infect a previously established SAMHD1 knockout mouse model and found that SAMHD1 inhibits the replication of MCMV in vivo. By comparing the replication of MCMV in vitro in myeloid cells and fibroblasts from SAMHD1-knockout and control mice, we found that the viral kinase M97 counteracts SAMHD1 after infection by phosphorylating the regulatory residue threonine 603. The phosphorylation of SAMHD1 in infected cells correlated with a reduced level of dNTP hydrolase activity and the loss of viral restriction. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and we identify the M97-mediated phosphorylation of SAMHD1 as a previously undescribed viral countermeasure.

Activation of E2F-dependent transcription by the mouse cytomegalovirus M117 protein affects the viral host range.

Cytomegaloviruses (CMVs) have a highly restricted host range as they replicate only in cells of their own or closely related species. To date, the molecular mechanisms underlying the CMV host restriction remain poorly understood. However, it has been shown that mouse cytomegalovirus (MCMV) can be adapted to human cells and that adaptation goes along with adaptive mutations in several viral genes. In this study, we identify MCMV M117 as a novel host range determinant. Mutations in this gene enable the virus to cross the species barrier and replicate in human RPE-1 cells. We show that the M117 protein is expressed with early kinetics, localizes to viral replication compartments, and contributes to the inhibition of cellular DNA synthesis. Mechanistically, M117 interacts with members of the E2F transcription factor family and induces E2F target gene expression in murine and human cells. While the N-terminal part of M117 mediates E2F interaction, the C-terminal part mediates self-interaction. Both parts are required for the activation of E2F-dependent transcription. We further show that M117 is dispensable for viral replication in cultured mouse fibroblasts and endothelial cells, but is required for colonization of mouse salivary glands in vivo. Conversely, inactivation of M117 or pharmacological inhibition of E2F facilitates MCMV replication in human RPE-1 cells, whereas replacement of M117 by adenovirus E4orf6/7, a known E2F activator, prevents it. These results indicate that E2F activation is detrimental for MCMV replication in human cells. In summary, this study identifies MCMV M117 as a novel E2F activator that functions as a host range determinant by precluding MCMV replication in human cells.

Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus.

Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus (HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection.

Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication.

The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production.

PUL21a-Cyclin A2 interaction is required to protect human cytomegalovirus-infected cells from the deleterious consequences of mitotic entry.

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.

Use of 5-ethynyl-2'-deoxyuridine labelling and flow cytometry to study cell cycle-dependent regulation of human cytomegalovirus gene expression.

The cell cycle position at the time of infection has a profound influence on human cytomegalovirus (HCMV) gene expression and therefore needs consideration in the design and control of HCMV experiments. While G0/G1 cells support the immediate onset of viral transcription, cells progressing through the S and G2 cell cycle phases prevent HCMV from entering the lytic replication cycle. Here, we provide two fast and reliable protocols that allow one to determine the cell cycle distribution of the designated host cells and monitor viral protein expression as a function of the cell cycle state. Both protocols make use of the thymidine analogue 5-ethynyl-2'-deoxyuridine and "click" chemistry to label HCMV-non-permissive S phase cells in a gentle and sensitive way.

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2-CDK-dependent sensor of the host cell cycle and differentiation state.

Upon cell entry, herpesviruses deliver a multitude of premade virion proteins to their hosts. The interplay between these incoming proteins and cell-specific regulatory factors dictates the outcome of infections at the cellular level. Here, we report a unique type of virion-host cell interaction that is essential for the cell cycle and differentiation state-dependent onset of human cytomegalovirus (HCMV) lytic gene expression. The major tegument 150-kDa phosphoprotein (pp150) of HCMV binds to cyclin A2 via a functional RXL/Cy motif resulting in its cyclin A2-dependent phosphorylation. Alanine substitution of the RXL/Cy motif prevents this interaction and allows the virus to fully escape the cyclin-dependent kinase (CDK)-mediated block of immediate early (IE) gene expression in S/G2 phase that normally restricts the onset of the HCMV replication cycle to G0/G1. Furthermore, the cyclin A2-CDK-pp150 axis is also involved in the establishment of HCMV quiescence in NTera2 cells, showing the importance of this molecular switch for differentiation state-dependent regulation of IE gene expression. Consistent with the known nucleocapsid-binding function of pp150, its RXL/Cy-dependent phosphorylation affects gene expression of the parental virion only, suggesting a cis-acting, virus particle-associated mechanism of control. The pp150 homologs of other primate and mammalian CMVs lack an RXL/Cy motif and accordingly even the nearest relative of HCMV, chimpanzee CMV, starts its lytic cycle in a cell cycle-independent manner. Thus, HCMV has evolved a molecular sensor for cyclin A2-CDK activity to restrict its IE gene expression program as a unique level of self-limitation and adaptation to its human host.

Inhibition of human cytomegalovirus immediate-early gene expression by cyclin A2-dependent kinase activity.

Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G(0)/G(1) phase of the cell division cycle. S/G(2) cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G(2)-specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G(1) was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies.

The latency-associated UL138 gene product of human cytomegalovirus sensitizes cells to tumor necrosis factor alpha (TNF-alpha) signaling by upregulating TNF-alpha receptor 1 cell surface expression.

Many viruses antagonize tumor necrosis factor alpha (TNF-α) signaling in order to counteract its antiviral properties. One way viruses achieve this goal is to reduce TNF-α receptor 1 (TNFR1) on the surface of infected cells. Such a mechanism is also employed by human cytomegalovirus (HCMV), as recently reported by others and us. On the other hand, TNF-α has also been shown to foster reactivation of HCMV from latency. By characterizing a new variant of HCMV AD169, we show here that TNFR1 downregulation by HCMV only becomes apparent upon infection of cells with HCMV strains lacking the so-called ULb' region. This region contains genes involved in regulating viral immune escape, cell tropism, or latency and is typically lost from laboratory strains but present in low-passage strains and clinical isolates. We further show that although ULb'-positive viruses also contain the TNFR1-antagonizing function, this activity is masked by a dominant TNFR1 upregulation mediated by the ULb' gene product UL138. Isolated expression of UL138 in the absence of viral infection upregulates TNFR1 surface expression and can rescue both TNFR1 reexpression and TNF-α responsiveness of cells infected with an HCMV mutant lacking the UL138-containing transcription unit. Given that the UL138 gene product is one of the few genes recognized to be expressed during HCMV latency and the known positive effects of TNF-α on viral reactivation, we suggest that via upregulating TNFR1 surface expression UL138 may sensitize latently infected cells to TNF-α-mediated reactivation of HCMV.

General blockade of human cytomegalovirus immediate-early mRNA expression in the S/G2 phase by a nuclear, Daxx- and PML-independent mechanism.

The onset of human cytomegalovirus (HCMV) lytic replication is strictly controlled by the host cell division cycle. Although viral entry of S/G2-phase cells is unperturbed expression of major immediate-early (MIE) genes IE1 and IE2 is tightly blocked in these cells. Besides the finding that cyclin-dependent kinase (CDK) activity is required for IE1/IE2 repression little is known about the nature of this cell cycle-dependent block. Here, we show that the block occurs after nuclear entry of viral DNA and prevents the accumulation of IE1/IE2 mRNAs, suggesting an inhibition of transcription. Remarkably, the presence of cis-regulatory regions of the MIE locus is neither sufficient nor necessary for IE1/IE2 repression in the S/G2 phase. Furthermore, the block of viral mRNA expression also affects other immediate-early transcribed regions, i.e. the US3 and UL36-38 gene loci. This suggests a mechanism of repression that acts in a general and not a gene-specific fashion. Such a nuclear, genome-wide repression of HCMV is typically mediated by the intrinsic immune defence at nuclear domain 10 (ND10) structures. However, we found that neither Daxx nor PML, the main players of ND10-based immunity, are required for the block to viral gene expression in the S/G2 phase. In addition, the viral tegument protein pp71 (pUL82), a major antagonist of the intrinsic immunity at pre-immediate-early times of infection, proved to be functional in S-phase cells. This suggests the existence of a yet undiscovered, CDK-dependent mechanism exerting higher-level control over immediate-early mRNA expression in HCMV-infected cells.

Cyclin-dependent kinase activity controls the onset of the HCMV lytic cycle.

The onset of human cytomegalovirus (HCMV) lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE) gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK) inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2) cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state.